introduction/background/objectives description of exact players involved sequential experimental...
TRANSCRIPT
“MOLECULAR CHAPERONE-MEDIATED RESCUE OF
MITOPHAGY BY A PARKIN RING1 DOMAIN MUTANT”
ROSE, JOHANNA M., NOVOSELOV, SERGEY S., ROBINSON, PHILIP A., AND CHEETHAM, MICHAEL E. HUMAN MOLECULAR GENETICS
VOL. 20 (2011): 16-27. PRINT.
PRESENTED BY: TREVOR BAUGHER
Outline
Introduction/Background/Objectives Description of Exact Players Involved Sequential Experimental Approaches
Testing Functions Under No Inducive Signal Proving Aggregates of Parkin are Protein Tests Showing Failure of RING1 Mutants to
Localize to Damaged Mitochondria Tests Confirming Parkin Induces
Autophagy/RING1 Domain Mutant Does Not Tests for Confirming Gain of Various Functions of
Parkin by Chaperone Protein Take Home Message Conclusion/Future Studies
Introduction/Background/Objectives Parkinson’s Disease
Common/Degenerative One Characteristic (inhibiting Parkin folding;
autophagy) Three Specific Domains
1 Ubl (N-term), 2 RING (C-term) RING1 Domain Governs Proper Function
RING1 Governs Proper Folding/Structure WT Parkin Can be Influenced by Uncouplers
carbonyl cyanide m-chlorophenylhydrazone (CCCP)
Aiming for Regain of Function by Chaperone Assist in Proper Folding/Aggregation Prevention HSJ1/1 J Domain (N-term), 2 UIM Domains (C-term)
Some of the Major Players FLAG-Parkin(WT)
FLAG-Parkin(R42P)
FLAG-Parkin(C289G)
HSJ1a(WT)
HSJ1a(H31Q)
HSJ1a(∆UIM)
Some Techniques of the Experimental Approaches
Fluorescence Microscopy
Staining GFP: Hsp70 (Parkin), LC3 (Autophagy Marker) MitoDsRed (mitochondria)
Gene Expression by Transfection SK-N-SH (neuroblastoma cells)
Experimental Approaches: Test 1: Testing Functions Under No Inducive Signal
Players GFP-Hsp70, Parkin(WT), Parkin(C289G),
Parkin(R42P) Reason for Test
Observe Normal Functions of Strains with no Treatment of Uncoupling Agents
Observe Aggregation Activity (inclusion bodies)
Results of Aggregation Parkin(WT)= 5% of cells Parkin(R42P)= 22% of cells Parkin(C289G)= 86% of cells
Test 2: Proving Aggregates of Parkin are Protein
Players GFP-Hsp70, Parkin(C289G)
Reason for Test Confirm That Aggregates Being Observed are
from Misfolded Protein
Results Inclusion Bodies Tested Positive for Hsp70
Test 3: Test Showing Failure of RING1 Mutants to Localize to Damaged Mitochondria
Players MitoDsRed, GFP-Hsp70, carbonyl cyanide m-
chlorophenylhydrazone(CCCP)(depolarization), Parkin(WT), Parkin(C289G), Parkin(R42P)
Reason for Test Observe Localization/Perinuclear Inclusions Observe Quantitative Overlap (% cells) Will Show Which Parkin Domain Governs
Function Results
Parkin(WT)= Significant Overlap(91% of cells) Parkin(R42P)= Significant Overlap(100% of
cells) Parkin(C289G)= Little Overlap(2% of cells)
Test 4: Tests Confirming Parkin Induces Autophagy/RING1 Domain Mutant Does Not Players
MitoDsRed, CCCP, GFP-LC3 (autophagy marker), Parkin(WT), Parkin(C289G)
Reason for Test Observe That RING1 Mutant Cannot Induce
Autophagy on its Own Observe Overlap Between Markers Shows That RING1 Domain is Responsible for
Autophagy (and Protein Misfolding) Results
Parkin(WT)= Significant Overlap Parkin(C289G)= Little to No Overlap RING1 Causes Inclusion Body Formation, Non-
localization and Now No Autophagy
Tests for Confirming Gain of Various Functions of Parkin by Chaperone Protein:
Test 5: HSJ1a Reduces Inclusion Body Formation of RING1
Players GFP-Hsp70, Parkin(C289G), HSJ1a(WT),
HSJ1a(H31Q), HSJ1a(∆UIM) Reason for Test
Observe the Incidence of Inclusion Bodies Determine Which Domain in HSJ1a Holds the
Function of the Chaperone Shows if HSJ1a Can Prevent Inclusion Bodies
Results HSJ1a(WT)/Parkin(C289G)= Significant Reduction
of Inclusion Bodies (from 81% to 17%) HSJ1a(H31Q)/Parkin(C289G)= No Reduction HSJ1a(∆UIM)/Parkin(C289G)= Significant Reduction
(from 81% to 27%)
Test 6: Test Effects of HSJ1a Variants on Restoring Localization of Parkin
Players GFP-Hsp70, MitoDsRed, CCCP, Parkin(WT),
Parkin(C289G), HSJ1a(WT), HSJ1a(H31Q), and HSJ1a(∆UIM)
Reason for Test Observe Localization of Parkin(C289G) to
Mitochondria Quantitate Overlap of Markers Shows if HSJ1a Can Restore Localization
Results Parkin(C289G)/HSJ1a(WT)= Significant Overlap Parkin(C289G)/HSJ1a(H31Q)= No Overlap Parkin(C289G)/HSJ1a(∆UIM)= Significant
Overlap
Test 7: Testing the Effects of HSJ1a on the Rescue of Autophagy
Players Red Fluorescent Hsp60, GFP-LC3, Parkin(WT),
Parkin(C289G), HSJ1a Reason for Test
Observe Induction of Autophagy by Parkin Quantitate Overlap of Markers Shows if HSJ1a Can Restore Autophagy
Results Parkin(WT)= Significant Overlap (68%) Parkin(C289G)= No Overlap Parkin(C289G)/HSJ1a(WT)= Some Overlap
(from 0% to 16%)
Take Home Message
HSJ1a Can Save Several Functions of the RING1 Domain Inclusion Bodies Relocalization Autophagy
J Domain in HSJ1a is Key Sheds Light on Important Information in
Prevention of Parkinson’s Disease Effects
Conclusion/Future Studies
HSJ1a(∆UIM) Always Functions at an Intermediate Level HSJ1a(WT)= Almost Full Restoration of Functions HSJ1a(∆UIM)= Restoration, But Not as Much HSJ1a(H31Q)= Minimal Restoration
UIM Domain, Possible Enhancer Conformational Change to More Induced Fit to
Parkin(C289G) Research
Replace UIM Domain With Other Functional Domain Invert UIM by Floxing/ See if This Decreases
Function of J Domain